Principal Investigator/Program Director (Last, First, Middle): MagneSS, R.R. Pjt. IV PI: GolOS, T.G. lines express a markedly different repertoire of genes, suggesting that these cell lines may respond differently to some experimental conditions, such as oxidative stress or adhesion and migration. Publications acknowledging support of HD38843 (collaborating P01P.l.s are in bold) Burleigh, D.W., K. Stewart, K.M. Grindle, R.R.Magness, H.H. Kay,and T.G.Golos. 2004 Proliferation and Apoptosis in Placenta from Diabetic Women. J. Soc. Gynecol. Invest. 11:36-41. Liu, Y.P.,O.V.Dovzhenko, M.A. Garthwaite, S.V.Dambaeva, M. Burning, LM. Pollastrini, and T.G. Golos. 2004 Maintenance of Pluripotency in Human Embryonic Stem Cells Stably Over-Expressing Enhanced Green Fluorescent Protein. Stem Cells Dev. 13:636-45. Liu, Y.P.,S.V.Dambaeva, Oksana V. Dovzhenko, M.A. Garthwaite, and T.G.Golos. 2005 Stable plasmid- based siRNA silencing of gene expression in human embryonic stem cells. Stem Cells Dev. 14:487-92. Burleigh, D.W., K. M. Grindle, R. L. Grendell, C.M. Kendziorski, R.R. Magness and T.G. Golos. 2006 Microarray Analysis of BeWo and JEG3 Trophoblast Cell Lines: Identification of Differentially Expressed Transcripts. In press. Golos, T.G., LM. Pollastrini, and B. Gerami-Naini. 2006. Human embryonic stem cells as a model for trophoblast differentiation. Sem. Reproof. Med. In press. Burleigh, D.W., K. M. Grindle, R. L. Grendell, C.M.Kendziorski, and T.G. Golos. Differential regulation of gene transcription by hyperglycemia in BeWo and JEG3 Trophoblast Cell Lines. In Preparation. Abstracts presented: D.W. Burleigh, K. Stewart, K.M. Grindle, R.R. Magness, H.H. Kay, and T.G. Golos. Proliferation and Apoptosis in Placenta from Diabetic Women. Presented at the 48th Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 14-17, 2001. D.W. Burleigh, M. Durning and T.G. Golos. Glucose-regulated placental and trophoblast (gene) mRNA expression. Presented at the 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, MD, July 28-31, 2002. Y. Song, Y.X. Wen, B. Gerami-Naini, M. Durning, T. G. Golos, and J. Zheng. Differentiation of Endothelial Cells from Human Embryonic Stem (ES)Cells. Presented at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, D.C., March 26-29, 2003. Burleigh, D.W., R.L.Grindell, K.M. Grindle, R.R.Magness and T.G.Golos. Microarray analysis of JEG3 and BeWo cell lines: comparison of differentially expressed transcripts. Presented at the 51st Annual Meeting of the Society for Gynecologic Investigation, Houston, TX, March 2004. Liu, Y.P., O.V. Dovzhenko, M.A. Garthwaite, S.V. Dambaeva, M. Durning, L.M. Pollastrini, and T.G. Golos. Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein. Presented at the 2nd Annual Meeting of the International Society for Stem Cell Research, Boston MA, June 2004. Gerami-Naini, B., O.V. Dovzhenko, M. Durning, M.A. Garthwaite, and T.G.Golos. Detecting expression of proliferation, differentiation and apoptosis markers in trophoblast cells derived from embryoid bodies grown in three-dimensional matrix structure and in suspension culture. Presented at the 3rd Annual Meeting of the International Society of Stem Cell Research, San Francisco, CA, June 23-25, 2005. Shift of focus to an ESC model for placental development. The control of trophoblast differentiation was a longstanding area of both interest and activity in our lab,in which we had used primary cultures as well as established human choriocarcinoma cell lines. In collaboration with James Thomson, we had previously shown that nonhuman primate ESC have an enhanced ability to differentiate into trophoblasts in comparison to mouse ESC (14, 15), an observation subsequently also determined for human ESC (13). However, this differentiation was still found to be of low efficiency in hESC. Thus, during this time period, we initiated independent studies in the laboratory to determine whether we could devise alternative experimental systems in which the ability to differentiate into trophoblasts was enhanced. Soon after we obtained results demonstrating the instructive effects of extracellular matrix on trophoblast differentiation (below), we recognized that the embryoid body paradigm represented an opportunity to develop a unique model for studying early events in placental morphogenesis, and we began to collaborate with Dr. Zheng in the P01 to develop the background data and the tools for the studies proposed in Projects //, III and IV. Thus, we are now focusing our efforts on the hESC paradigms we have developed for modeling placental morphogenesis and PHS 398/2590 (Rev.09/04) IV-21 Continuation Format Page Principal Investigator/Program Director(Last, First, Middle): MagneSS, R.R. Pjt. IV PI: GoloS, T.G. this will be the focus of the competitive renewal of this Project. It should be recognized, thus, that Project IV in the preceding funding period supported in part experiments that collected the preliminary data for Core B, Project IV, andProject III, andnewdirections undertaken by Project II in this A1submission. MG3 Every day Every 3 days Every 5 days Every day Every 3 days Every Sdays Time of EB Culture o 10 20 so Day of Culture Fig. 3. Hormone secretion by EBs during 144 hrs of Fig. 4. Secretionof hCG by Matrigel-embedded EBs. The suspension culture, in comparison with unconditioned results of three representative experiments with ~ 50 medium. EBs/3ml dish are shown. Embryoid body formation. Although hCG secretion is detectable in spontaneously differentiating ESC cultures (18,and Gerami and Golos, unpublished), the typically modest levels of hCG secretion indicate that spontaneous trophoblast differentiation, while consistently detectable, is not extensive. We considered embryoid bodies (EBs) as an alternative model to evaluate trophoblast differentiation from hESC. EBs were prepared generally as previously described (32). Within 2 days after the initiation of suspension EB culture, the secretion of hCG, progesterone and estradiol was detectable in the culture medium, in comparison with unconditioned medium (Fig. 3). Reasoning that the EBs may be a good model for not only trophoblast differentiation, but also perhaps placenta! morphogenesis, we initiated pilot studies to evaluate EB cell growth in an ECMenvironment as a first approximation of implantation in the uterine endometrium. We cultured EBs in suspension for 9 days, and transferred them individually to 50-100 ul volume drops of growth factor reduced (BD Biosciences) Matrigel which had been allowed to gel at 37[unreadable]C. The Matrigel "rafts" containing the EBs were then transferred to fresh PHS 398/2590 (Rev. 09/04) IV-22 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Magness, R.R. Pjt. IV PI: GolOS, T.G. EB culture medium. Culture was then continued, with gentle rocking,to prevent adhesion to the plastic. Please see Core B for details on embryoid body formation and culture in Matrigel. Fig. 5. Trophoblast-like outgrowths projecting from the surfaces of representative EBs explanted to Matrigel rafts. With these cultures, we noted a frequent appearance of projections from the surface of the EBs (Fig. 5). Although the appearance of the outgrowths was heterogeneous, virtually all EBs exhibited outgrowths into the Matrigel environment. These Matrigel-embedded EBs were maintained in culture for up to 50 days, and we determined hCG secretion into the culture medium (Fig. 4). Replicate cultures had medium replaced every 1, 3 or 5 days. While hCG secretion was detectable but relatively modest for the first 20 days in culture, there was a dramatic increase in hCG secretion that occurred reproducibly at approximately day 20 (three independent experiments are shown in Fig. 4). High levels of hCG (and progesterone and estradiol-17p) secretion were sustained for at least 4 weeks, and daily medium change promoted the highest level of hCG secretion. Additional experiments with delayed transfer to Matrigel demonstrated that exposure to matrix and not age of the EB was the critical element in the initiation of trophoblast differentiation (Appendix). Comparing secretion rates in suspension vs. Matrigel culture, an increase of >10,000-fold induction is typical. Thus, the dramatic increase in hormone secretion following Matrigel explantation suggests that in suspension EBs, trophoblasts are relatively undifferentiated but that Matrigel exposure promotes cells from a progenitor/stem trophoblast, to a committed cell undergoing terminal differentiation. IHC studies demonstrate readily discerned populations of cytokeratin/hCG-positive cells. We conclude that trophoblast-like cells differentiate in EB cultures. Given these results, we wished to determine if association of EBs with Matrigel under "two-dimensional" conditions was sufficient to promote long-term hormone secretion. This is particularly important with regards to our ability to evaluate differentiation on diverse matrix surfaces. We conducted experiments with suspension EB cultures transferred to culture dishes and allowed to adhere to a thin coating of Matrigel on the culture surface (Fig. 6, also Appendix). . EB plated on Matrigel coated plates 1200 1000 E 800 [ I O 600 [[unreadable] 400 . 200 'T J A n n n fi H III 1 5 10 11 12 13 14 15 16 17 18 19 20 21 22 25 Day of Culture Fig. 6. Secretion of hCG in EBs allowed to Matrigel-coated dishes in adherent culture. PHS 398/2590 (Rev. 09/04) L f - 30 35 40 43 attach to Fig. 7. Representative images of cells derived from embryoid bodies cultured on Matrigel. Syncytial cells were seen 6 days after passage, whereas freshly passaged cells were all mononuclear(not shown). IV-23 Continuation Format Page Principal Investigator/ProgramDirector (Last, First, Middle): MagneSS, R.R. Pjt.IV PI: GolOS, T.G. The results demonstratethat hCG, progesteroneand estradiol-17p secretion beganto increaseapproximately 10 days after attachmentto the culture surface and was sustainedfor 2-4 weeks, depending on the thickness ofthe Matrigel. For several of the replicate wells in this experiment, we wished to determine if the outgrowths could be passaged and replated and would sustain proliferation. Cellular outgrowths were resuspended following trypsin treatment and were replated on Matrigel-coated plates. Cells were readily adherent and continued to proliferate, and passagedcells in all wells exhibited at least some syncytiumformation (Fig. 7), characteristic of both primary human villous cytotrophoblast cultures as well as BMP4-treated hES cells (16,33). In addition, hCG, progesterone and estradiol secretion was maintained through at least two passageswith these cells (not shown). Similar results have been obtained in experiments with cytokeratin-positive cells dispersed from suspension EBs with limitedtrypsin digestion. EB outgrowths on individual ECMcomponents. Wehave begun to conduct preliminary experiments onthe differentiation of trophoblastic outgrowths from hESC-derived embryoid bodies, as assayed by hCG secretion. Initial results are shown in Fig. 8, which presents hCG levels in EBs plated in 2-dimensional culture on Matrigel, laminin, and poly-l-lysine-coated dishes. While culture on Matrigel promotes the highest secretion of hCG, laminin (Fig. 8) and collagen IV (not shown) also promote substantially higher levels than other surfaces, includingpoly-l-lysine. Hormone secretion is not due to substantially different EB adhesion (cell number, day2) or proliferation (cell number, day 24) (Fig.9). Further 2-D model studies, extended to signaling and microarray analysis, are proposed in Aim 1. Fig. 8. Secretion ofhCGin 2-DEBcultures on varied Fig. 9. Cellnumber in 2-DEBculture experiments on matrix protein surfaces. various matrix surfaces. Integrin signaling components. Relevant to Specific Aim 1,we have conducted preliminary western blot and immunohistochemistry analysis of undifferentiated hESC and suspension and 2-D Matrigel cultured EBs from hESC. Western blots were optimized by the Molecular Core. Preliminary data in Fig. 10 demonstrates expression of several of these molecules by western blot of extracts from suspension EBs, including FAK, ROCK, and paxillin. While not unexpectedly, all are expressed in differentiated as well as undifferentiated cells, defining their activation/phosphorylation states is the critical experiment, which is proposed in